Method for obtaining a Raman spectrum of a sample or particle

ABSTRACT

A micro-fluidic system comprising means for optically trapping a particle and a Raman excitation source for causing Raman scatter from the particle whilst it is in the optical trap.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/303,526, filed Dec. 4, 2008, entitled: RAMAN SPECTROSCOPY, which,in turn, is a national stage application (filed under 35 § U.S.C. 371)of PCT/GB2007/002121, filed Jun. 8, 2007 of the same title, which, inturn, claims priority to Great Britain Application No. 0611289.0, filedJun. 8, 2006 of the same title; the contents of each of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to Raman spectroscopy. In particular, theinvention relates to the use of Raman spectroscopy for investigatingbiological material, for example, single cells.

BACKGROUND OF THE INVENTION

Raman spectroscopy is a powerful technique that relies on collection ofinelastically scattered laser light from a sample. This light exhibits afrequency shift that reflects the energy of specific molecularvibrations within the sample of interest. Hence, it can provide adetailed chemical composition of the sample, i.e. a chemicalfingerprint. The technique has wide potential in biomedical science asit may be applied to samples over a wide size range from single cellsthrough to intact tissue.

One of the major challenges of Raman spectroscopy is the inherently weaknature of the signal. In addition, a Raman signal may be obtained fromthe local environment surrounding the sample, typically making itdifficult to discern the molecular signatures of interest. Thus,considerable effort has focused on enhancing the ratio of signal tobackground noise. By increasing the acquisition time to several minutes,the signal to noise ratio can be improved. However, in someenvironments, long acquisition times can cause damage due to extendedirradiation by the excitation laser and the mechanism required to holdthe particles under investigation in the measurement position. These areparticular problems when investigating live cells or tissue samples.

Some solutions to the problems with conventional Raman spectroscopy havebeen proposed. Many of these involve the inclusion of additionalmaterial, for example nano-particles, in the samples that are beinginvestigated. However, this is not ideal for the investigation of wholecells as the precise positional control of the foreign particles isdifficult. Additionally, the enhancement achieved with the use offoreign particles is confined to the immediate surface of the particles(˜10 nm) making the measurement of the overall Raman signal impossible.One technique that does not require the addition of foreign particlesuses wavelength modulation. This is described in the article“Wavelength-Modulation Raman Spectroscopy” by Levin et al, Appl. PhysLetter 33(39), 1 Nov. 1978. This technique increases the sensitivity ofa Raman spectroscopic system by modulating the wavelength of theexcitation light, and then using this to distinguish the sample's Ramanresponse from background radiation and/or noise. The system describeduses a tuneable dye laser and single channel slowly scanning detection.A problem with this is that the scan takes about 50 minutes for thewhole spectra. Additionally the method relies on very large, expensiveoptics and is inappropriate for many practical applications, inparticular the investigation of single cells.

One of the most promising areas of application for Raman spectroscopy isin the discrimination between sets of biomedical samples e.g. cancerdiagnostics. Here, it is advantageous to have short acquisition times,especially if a live patient rather than a retrieved sample is beingstudied. It is also important to reduce the impact of fluorescence, asthis has a high patient to patient and even cell to cell variabilitythat can heavily reduce the performance of any subsequent diagnosticmodels. One of the most widely used tools for discriminating between theRaman spectra acquired from sets of biomedical samples is PrincipalComponent Analysis (PCA).

Principal components analysis (PCA) is a statistical technique used tochange the representation of a multidimensional data set. A newrepresentation or coordinate system is constructed such that thevariance of the data sets is biggest for the first coordinate componentof the new representation. This is then called the first principalcomponent. The second biggest variation lies the on the secondcoordinate of the new representation, and so on. Finally, the data setdimension is reduced by retaining only the first few principalcomponents that account for most of the variance of the original dataset. It is these low-order components that often contain the “mostimportant” aspects of the data set. Using PCA to examine Raman spectrafrom sets of biomedical samples allows combinations of Raman peakfluctuations to be found that can then be used to discriminate betweenthe Raman spectra from the sets of biomedical samples.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a micro-fluidicsystem comprising means for optically trapping a particle and means forobtaining a Raman spectrum from the particle whilst it is in the opticaltrap.

Typically, the means for forming an optical trap comprise a dual beamarrangement, in which counter propagating optical beams are used to holdthe particle. Because the trapping beams are divergent, this arrangementreduces the chance of damage to the particle under investigation. Thisis particularly advantageous when the particle is a cell. The laser forexciting the Raman scatter may be placed orthogonal to the trappingbeams.

Means may be provided for modulating the Raman excitation signal. Themodulation means may be operable to encode information onto one or moreparameters of the excitation signal. The modulation means may beoperable to modulate one or more of the excitation laser drivingcurrent; intra-cavity or external cavity grating position and/ororientation; change of the cavity length, using, for example mechanicalor opto-electric means; polarization variation; excitation modevariation and variation of the optical properties of any intra-cavity orexternal cavity non-linear optical elements.

Any suitable laser can be used to form the Raman excitation signal,although a laser diode in a Littrow or Littman-Metcalf configuration ispreferred. Alternatively, two or more laser sources may be combinedwhere each has a different wavelength. In this case, each of the sourcescan be independently switched and its intensity varied to achieve anefficient modulated multi wavelength excitation.

The Raman excitation can also be provided by a broadband light sourcesuch as mode-locked pulsed lasers, delivering 100 fs pulses, forexample, or other sources such as a white light source. These sourcescan have their spectral phase/chirp specially engineered and/ormodulated. This can be achieved, for example, by passing the pulsethrough a Fabry-Perot resonator giving a periodic spectral phasemodulation. More complex spectral phase/chirp modulation can be obtainedthrough the use of a Spatial Light Modulator (SLM) in conjunction withsome spectral dispersion elements such as prisms or other photonicdevices.

Means may be provided for doing a principal component analysis. Inaccordance with the invention, a single modulated measure from a cellconsists of multiple, short duration, spectra taken with the excitationlaser at different wavelength. All the spectra together form a data seton which a principal component analysis can be performed. Contrary toconventional PCA, the value of the first principal component is not ofinterest. Instead, it is the associated Eigen-spectra, which is thebasis vector associated with the first principal component. It is theseEigen-spectra (basis-vector) that are then the differential spectra.Here, the PCA is not used to reduce the dimensionality of the data setbut to extract the element with the largest variation.

According to the present invention, there is a method for obtaining aRaman spectrum comprising exciting a sample using radiation; capturinglight emitted from the sample; modulating the excitation radiation;capturing light emitted in response to the modulated radiation and usingthe captured radiation to obtain the Raman spectrum. Preferably, thescattered radiation is captured using a multi-channel spectrometer,ideally a CCD camera.

The method may further involve correlating modulations in the excitationradiation with variations in the captured spectra. By doing this, theRaman peak can be more accurately identified, as backgroundfluorescence, for example, should not vary with changes in theexcitation signal.

By analyzing the light emitted in response to both the initialexcitation radiation and its modulated version using PCA, furtherimprovements may be made. This provides a simple technique for pullingout variations in the acquired spectra. If the modulated spectra are fedinto a PCA routine, this will look at the variation in the spectra.Because of the modulation, this variation is the moving Raman spectrumonly, as the fluorescence remains steady. Thus the PCA routine outputs aspectrum, or principal component that is the differential Raman spectrumof the sample. For the extraction of the differential Raman signal aminimum of one modulation period is necessary.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the invention will now be described by way of exampleonly and with reference to the accompanying drawings, of which:

FIG. 1A is an image of a polymer microsphere that is optically trappedin a micro-fluidic channel and exposed to Raman excitation, togetherwith the resultant Raman spectra;

FIG. 1B is an image of an HL60 cell that is optically trapped in amicro-fluidic channel and exposed to Raman excitation, together with theresultant Raman spectra;

FIG. 2A is a plot of laser intensity as a function of time;

FIG. 2B is a plot of laser wavelength as a function of time;

FIG. 3 is a complete Raman spectrum including background, noise,excitation and Raman resonance peaks;

FIG. 4 is a plot of the variance of 90 Raman Spectra of 0.5 s each;

FIG. 5 shows the average Raman spectra using the wavelength tracking andsignal renormalization method;

FIG. 6A is a plot of wavenumber versus intensity of the laser;

FIG. 6B is a plot of binned and averaged spectra;

FIG. 7 is a plot of integrated differential Raman signal as a functionwavelength;

FIG. 8 is plot of simulated wavelength versus intensity in amulti-stable lasing device;

FIG. 9 is a diagram of an alternative optical arrangement for trapping asingle cell and obtaining Raman spectra from it;

FIG. 10 is a single Raman spectrum recorded at one wavelength;

FIG. 11 is a sequential recording of the Raman signal as the laser, andhence Raman spectra, was modulated between two fixed wavelengths;

FIG. 12 is a plot of a differential Raman spectrum of a cell extractedfrom the modulated Raman signal, and

FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D show the results of a PCAanalysis carried out to compare the effect of acquiring a Raman signalusing conventional processes and a process in accordance with theinvention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1A and FIG. 1B show a microfluidic device 110 that is operable toform an optical trap in a micro-fluidic channel. Any suitable means canbe used for causing fluid to flow through the device. The optical trapis formed using two counter propagating diverging beams. In thisexample, the counter propagating beams are provided via two opticalfibers that are positioned on opposing sides of the micro-fluidicchannel. In this case, the channel is a micro-capillary. Radiation isdirected via the fibers into the micro-fluidic channel, so that cells orother particles within the fluid can be trapped. Optical traps can beused to allow micrometer-sized particles to be held, moved and generallymanipulated without any physical contact. This has been well documented,for example see Ashkin et al Optics Letters Vol. 11, p 288 (1986).Orthogonal to the fiber ends (not shown) is an objective lens fordirecting a Raman excitation beam onto the cell and capturing theemitted signal so that it can be recorded.

To test the arrangement of FIG. 1A and FIG. 1B, a flow system consistingof a capillary tube 102, of square cross-section size 80 microns, wasconnected to a syringe or gravity feed pump. Initially, 10 micronpolymer particles were flowed through the capillary tube 102, andtrapped using the counter propagating beams, as and when desired. A 50mW Raman examination beam was then introduced from below using a Nikon×50 NA 0.9 oil immersion. FIG. 1A shows a polymer microsphere 100trapped inside the capillary tube 102, together with the spectra 105obtained from the polymer microsphere 100. FIG. 1B shows an HL60 cell101 trapped inside the capillary tube 102, together with the spectra 106obtained from the HL60 cell 101.

By using optical trapping in a microfluidic environment, damage to theparticle/cell that is under investigation can be minimized. However, tofurther reduce this, a statistical approach can be used to allow theRaman signals to be recorded very rapidly from a single cell. Thismethod relies on modulation of the excitation laser, and in particulartuning of the laser wavelength. This can be done using continuous ordiscontinuous tuning. Statistical analysis of the resultant Ramanscatter allows a significant reduction in the time needed to record thesignals. This can be done without the addition of foreign particles,such as nanoparticles, specialist surfaces, and/or enhancement schemes.

The physical properties, such as wavelength and intensity, of the Ramanexcitation vary in time. The resulting Raman signal is then also subjectto variations but in a complex way. Indeed, depending on their physicalorigin the different parts of the Raman spectra behave differently. Ifthe wavelength is modulated then the Raman peaks in the spectra incur ashift in wavelength while the fluorescence background remains constant.In the case of amplitude variation, both peaks and background change inamplitude.

The method of the present invention uses a general wavelength, frequencyand amplitude or other parameters variation of the excitation andcorrelates this with the measured Raman spectra to distinguish betweenthe different components of the spectrum, i.e. background, Raman peaksand noise. The input excitation is encoded with a variation which thenis decoded at detection time distinguishing thus between signal, noiseand background. Variation of the parameters is used to quantify thecorrelated variation of the Raman signal.

The encoding method is based on the variation of controlling theparameters of the Raman excitation source such as the laser or anydevice delivering the necessary excitation output. Examples of theseparameters are: laser, diode or device driving current; intra-cavity orexternal cavity grating position and/or orientation; mechanical oropto-electric change of the cavity length; polarization variation;excitation mode variation and variation of the optical properties of anyintra-cavity or external cavity non-linear optical elements.

Another way to achieve source variation is by using bistable or multistable lasers that naturally oscillate in a controlled or chaoticfashion between different wavelength and states. Alternatively, two ormore laser sources can be combined where each has a differentwavelength. Each of the sources can be independently switched and itsintensity varied to achieve an efficient modulated multi wavelengthexcitation.

FIG. 2A and FIG. 2B show the effect of varying the driving current ofthe diode laser device. FIG. 2A is a plot of laser intensity as afunction of time, and FIG. 2B is a plot of laser wavelength as afunction of time. As can be seen, varying the drive current induces awavelength shift and an excitation intensity variation. Because of thenon-linear properties of the laser, discrete wavelength jumps occur asthe current is varied. These jumps correspond to laser mode hopping.

To obtain the sample response, Raman spectra are repeatedly acquired inas short as possible time slots whose duration is related to the speedof variation of the excitation parameters. Over this duration, theexcitation parameters should not vary. For practical reasons, thespectral snapshot can also contain the excitation spectra suitablyattenuated in intensity. The excitation spectral information such aswavelength, amplitude and bandwidth can then be retrieved from thissnapshot. Alternatively other measures can be used to deduce theexcitation characteristics and their variation or the variation can belinked to the control parameters after suitable calibration. Everysnapshot is then stored together with the excitation parameters forreal-time or successive data treatment.

FIG. 3 shows a Raman spectrum that was acquired with duration of 0.5 s.The excitation parameters can be retrieved from the furthest left peak(A), which corresponds to the excitation laser. There are multiple waysto retrieve the Raman peaks from a family of short scans, each taken fordifferent excitation parameters. Some methods cancel directly thebackground while others do not. A non-exhaustive list of possiblemethods includes statistical post processing (variance), real time/postprocessing signal tracking (spectral lock-in amplifier), and realtime/post processing leading to differential signal (statisticalapproach).

Statistical post processing involves looking at the variation of afamily of spectra as a function of wavelength. If the excitationwavelength variation is large enough then the variance of the family ofspectra will show different levels of variance for the noise, backgroundand Raman peaks. Indeed the shift of the excitation wavelength implies ashift of the peaks, which is equivalent to a large intensity variationat a given wavelength. The variance of the peak will thus be much higherthan the variance of the surrounding region. FIG. 4 shows the resultingRaman spectra after using the statistical post processing method thatcalculates the variance from 90 Raman spectra of 0.5 s each.

Real time/post processing signal tracking (spectral lock-in amplifier)involves using the amplitude and wavelength position of the excitationlaser peak to shift and normalize the individual 0.5 s Raman spectrabefore averaging them. However, this method does not cancel thebackground and is disadvantaged by the laser mode hopping. It is similarto a lock-in amplifier as it locks-in onto the reference excitationwavelength and uses its shift to reconstruct the resonances. FIG. 5shows the processed Raman spectra using the excitation wavelength andamplitude tracking method.

Real time/Post processing leading to differential signal (statisticalapproach) involves using a differential signal to eliminate thebackground. This can be achieved by using two laser states withdifferent wavelengths. When plotting the amplitude versus the wavelengthof the excitation laser while the driving currant is varied the numberof modes accessed by the parameter variation can be recognized, as shownin FIG. 6A, which is a plot of wavenumber versus intensity of the laser,and FIG. 6B, which is a plot of binned and averaged spectra. In thiscase, the wavelength position of the laser peak is used to average onlyspectra where the excitation laser has a specific wavelength. Thespectra in a bin are normalized with the amplitude of the laserintensity and then averaged. Because of the bi-stability there are onlytwo bins. The differential signal corresponds in this case to thedifference between the red and blue curve. When calculating thisdifference the background part of the signal is removed. The differencecan then be integrated to retrieve original Raman resonance peaks, asshown in FIG. 7. This method can be generalized to multi stable lasingdevices. FIG. 8 shows a simulated wavelength versus intensity in amulti-stable lasing device. In this multiple stabilities will increasethe differential signal as this can be calculated using n-pointdifferential formulas.

FIG. 9 shows a more detailed system for providing a modulated Ramanexcitation signal in accordance with the invention. This has a laser 900that can be modulated in some form: mechanically, optically or bycurrent. This is then reflected against a holographic notch filter 901that reflects a very narrow band around the wavelength and transmits allother wavelengths, into a microscope objective 902 that focuses the beamto the sample. The Raman signal is collected by the same microscopeobjective 902 and transmitted through the notch filter 901 onto adichroic mirror 903, which reflects the infrared Raman scatter whilstallowing the visible incoherent light, which illuminates the sample, topass to a viewing camera 904. This allows an image of the sample understudy to be collected as well as its Raman spectrum. The collected Ramanscatter is then passed through an optional confocal aperture 905 toreject any unwanted signal surrounding the sample of interest. Thesignal is finally imaged onto a 550 mm spectrograph 906 equipped with a300 lines/mm grating to separate spatially all the collected Ramanwavelengths that are imaged onto a multi-channel detector, for example aCCD camera 907. The CCD camera 907 is a liquid nitrogen cooled CCD thathas a pixel array of 2048×512 with each pixel measuring 13.5 μm square,the array having a bandwidth of one pixel, i.e. about 0.15 nm. Thecombined resolution of the spectrograph is 0.078 nm allowing themovement of the laser and hence Raman spectrum to be captured.

In order to remove or reduce fluorescence in the acquired Raman spectra,as well as reduce the acquisition times the excitation wavelength ismodulated and multiple spectra collected. The Raman spectra are thenextracted from these multiple spectra. To improve extraction of theRaman spectrum from the modulated data, an external cavity laser diodewas used in a Littman-Metcalf configuration. This configuration allows asignificantly greater tuning range (˜30 nm) compared to the bandwidth ofone pixel (0.15 nm) of the detecting CCD mounted on the spectrometer,improving the detection of the modulation significantly. This laser wasused to switch between two wavelength positions that in turn modulatedthe Raman spectra between two positions. A signal was acquired at eachwavelength position as it was moved between the two wavelengths. Asingle spectrum can be seen in FIG. 10, which shows single spectrumrecorded at one wavelength position. Multiple signals were acquired ateach wavelength position as it was modulated. FIG. 11 shows thesequential recording of the Raman spectra as the laser was modulatedbetween two fixed wavelengths. The jumps in the spectra can be clearlyseen. The laser is on the extreme left and the Raman peaks to the rightof this.

To improve the detection of variations in the acquired spectra amodified version of conventional PCA can be used. This pulls outvariation in the acquired spectra. If the modulated spectra are fed intoa PCA routine, this will look at the variation in the spectra. Becauseof the modulation, this variation is the moving Raman spectrum only, asthe fluorescence remains steady. Thus the PCA routine outputs aspectrum, or principal component that is the differential Raman spectrumof the sample. For the extraction of the differential Raman signal aminimum of one modulation period is necessary.

FIG. 12 shows an example of a differential spectrum after PCAprocessing. This differential spectrum can be integrated to reproducethe normal Raman spectrum of the sample or left as is for furtherstatistical analysis. An advantage of using PCA in this way is that theoutput is the variation in the Raman spectrum. Thus, there is no need totrack the laser line allowing points of interest in the spectra to beidentified and giving much more flexibility in the choice ofinstrumentation such as which grating to use. This method also removesthe fluorescence background. It should be noted that fluorescence is notalways a problem in viewing the spectrum, but is more of a problem insubsequent statistical analysis where it can severely affect theefficiency of discrimination between two sample sets such as healthy anddiseased cells.

In order to evaluate the ability of this technique to effectively removefluorescence and potentially reduce acquisition times a comparison wasmade with conventional PCA Raman processing and the combinedmodulation/PCA processing of the invention. This was done for sets ofRaman spectra acquired from different regions in a biological cell,nucleus and cytoplasm. Ten Raman spectra were collected from the nucleusand cytoplasm. The spectra were acquired in two minutes for bothconventional PCA Raman processing and the combined modulation/PCAprocessing of the invention. To test the acquisition time reducingpotential of the invention spectra for the modulated/PCA were alsoacquired in one minute.

FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D show the results of the PCAanalysis carried out to compare the effect of acquiring the signalnormally to the modulated/PCA method of acquiring the Raman signal. FIG.13A shows the diagrammatic definition of the resolution used in FIG.13B, FIG. 13C, and FIG. 13D. From FIG. 13B and FIG. 13C, it can be seenthat the resolution greatly increases when the Raman signal is acquiredusing the modulated/PCA method. This is because it removes thefluorescence that has a negative impact on the diagnostic PCA model.This may be important in medical diagnostics as patient-to-patientvariability in fluorescence may greatly affect any diagnostic modelsbased on Raman spectroscopy. Furthermore, as shown in FIG. 13C and FIG.13D even when the acquisition time is halved, the modulated/PCA Ramanspectra provides a much better resolution compared to the discriminationbased on the normal acquisition indicating that the acquisition timecould be reduced by a factor of at least two.

The present invention provides a system that allows single cells to beoptically trapped and held, and Raman signals to be acquired from thesecells in a very short time. Contrary to 1978 paper, where the Ramansignal was acquired with a slowly scanning single channel detectionsystem (2.4 nm/min), the present invention combines the advantages ofacquiring the modulated Raman signals with modern multi-channel CCDdetection allowing a rapid acquisition whilst excluding the fluorescencebackground. Additionally, the invention improves subsequent statisticalanalyses such as Principal Component Analysis (PCA) important medicaldiagnostics for example. Using excitation signal modulation, signals canbe acquired in ˜ 1/10 to 1/50 of the time that would normally berequired. This means that damage to cells due to over exposure to theRaman excitation can be minimized. A skilled person will appreciate thatvariations of the disclosed arrangements are possible without departingfrom the invention. For example, whilst a micro-capillary is describedin other embodiments, the microfluidic flow may be implemented usingchannels made using soft lithography in PDMS or similar and the size ofthe channel may naturally vary. Accordingly the above description of thespecific embodiment is made by way of example only and not for thepurposes of limitation. It will be clear to the skilled person thatminor modifications may be made without significant changes to theoperation described.

The invention claimed is:
 1. A method for obtaining a Raman spectrumcomprising: exciting a sample or particle using first excitationradiation at a first excitation wavelength to cause emission of a firstRaman signal; capturing first light scattered from the sample orparticle when exciting the sample or particle using the first excitationradiation; modulating the first excitation radiation to create secondexcitation radiation at a second excitation wavelength; exciting thesample or particle using the second excitation radiation to causeemission of a second Raman signal; capturing second light scattered fromthe sample or particle when exciting the sample or particle using thesecond excitation radiation; forming a data set using the capturedscattered first light and the captured scattered second light; andperforming principal component analysis on the data set to identify adifferential Raman signal or a function thereof for the sample orparticle, wherein the first and second excitation wavelengths aredifferent from each other, and wherein the method does not requireknowledge of any relationship between the first and second excitationwavelengths other than a knowledge that the first and second excitationwavelengths are different from each other.
 2. A method as claimed inclaim 1 further comprising: repeating at least once the following steps:modulating the first excitation radiation to create further excitationradiation at a further excitation wavelength; exciting the sample orparticle using the further excitation radiation to cause emission of afurther Raman signal; and capturing further light scattered from thesample or particle when exciting the sample or particle using thefurther excitation radiation, wherein forming the data set comprisesforming the data set using the captured scattered further light, whereinthe first, second and further excitation wavelengths are different fromeach other, and wherein the method does not require knowledge of anyrelationship between the first, second and further excitationwavelengths other than a knowledge that the first, second and furtherexcitation wavelengths are different from each other.
 3. A method asclaimed in claim 1, wherein said exciting a sample or particle using thefirst excitation radiation to cause emission of a Raman signalcomprises: optically trapping the sample or particle by forming a dualbeam arrangement, in which counter propagating optical beams are used tohold the particle; and emitting the first excitation radiation from aradiation source orthogonal to the trapping beams, wherein the radiationsource comprises two or more laser sources each independently switchableand operable to vary its intensity between multiple levels, each of themultiple levels of intensity being sufficient to cause Raman scatter,thereby to achieve intensity modulated multi wavelength excitationradiation.
 4. A method as claimed in claim 3 further comprisingmodulating the first excitation radiation emitted from the two or morelaser sources.
 5. A method as claimed in claim 4 further comprisingencoding information onto one or more parameters of the first excitationradiation.
 6. A method as claimed in claim 1, wherein modulating thefirst excitation radiation comprises modulating one or more of thefollowing: excitation laser wavelength; excitation laser drivingcurrent; intra-cavity or external cavity grating position and/ororientation; laser cavity length; excitation laser polarization;excitation mode and optical properties of any intra-cavity or externalcavity non-linear optical elements.
 7. A method as claimed in claim 1comprising modulating the first excitation radiation by switchingbetween two or more different wavelengths.
 8. A method as claimed inclaim 1, wherein the first excitation radiation has a first excitationintensity, wherein the second excitation radiation has a secondexcitation intensity, and wherein the first and second excitationintensities are different from each other.
 9. A method as claimed inclaim 8, wherein the method does not require knowledge of anyrelationship between the first and second excitation wavelengths and thefirst and second excitation intensities other than a knowledge that thefirst and second excitation wavelengths are different from each otherand a knowledge that the first and second excitation intensities aredifferent from each other.